Influenza vaccine

ABSTRACT

The present invention relates to influenza vaccines for human and veterinary use. In particular, the present invention provides a vaccine able to effect long term and cross-strain protection by including at least two influenza virus epitopes expressed as a chimeric polypeptide wherein at least one epitope is influenza A virus matrix protein epitope and the second epitope is a haemagglutinin peptide epitope.

This application is a 371 filing of International Patent ApplicationPCT/IL2006/001403 filed Dec. 6, 2006, which claims the benefit ofprovisional application Nos. 60/742,574 and 60/742,530, each filed Dec.6, 2005.

FIELD OF THE INVENTION

The present invention relates generally to influenza vaccines for humanand veterinary use. In particular, the present invention provides avaccine able to elicit long term and cross-strain protection comprisinga plurality of chimeric proteins comprising at least two influenza viruspeptide epitopes wherein at least one epitope is an influenza A virusmatrix protein (M) peptide epitope and the second epitope is ahaemagglutinin (HA) peptide epitope. Particularly advantageous epitopesinclude M1 or M2 N-terminus peptide epitopes and an influenza A orinfluenza B B-cell type HA epitope.

BACKGROUND OF THE INVENTION Influenza

Influenza is a disease caused by viruses of three main subtypes,Influenza A, B and C, which are classified according to their antigenicdeterminants. The influenza virion consists of a single stranded RNAgenome closely associated with a nucleoprotein (NP) and enclosed by alipoprotein envelope lined by matrix protein (M1) and carrying two majorsurface glycoprotein antigens, haemagglutinin (HA) and neuraminidase(NA). The HA and NA glycoproteins are most susceptible to change; forexample, there are 16 immune classes of HA and 9 different NA classesthat provide the basis for the different influenza virus subtypes likeH1N1 or H3N2. Influenza A virus has an additional transmembraneglycoprotein, M2, which is highly conserved between the different HNsubtypes. The M2 gene encodes a protein having 96-97-amino-acids that isexpressed as a tetramer on the virion cell surface. It is composed ofabout 24 extracellular amino acids, about 19 transmembrane amino acids,and about 54 cytoplasmic residues (Lamb et al, 1985).

Influenza A and B viruses are the most common causes of influenza inman. Influenza has an enormous impact on public health with severeeconomic implications in addition to the devastating health problems,including morbidity and even mortality. Infection may be mild, moderateor severe, ranging from asymptomatic through mild upper respiratoryinfection and tracheobronchitis to a severe, occasionally lethal, viralpneumonia.

Influenza viruses have two important immunological characteristics thatpresent a challenge to vaccine preparation. The first concerns geneticchanges that occur in the surface glycoproteins every few years,referred to as “antigenic drift”. This antigenic change produces virusesthat elude resistance elicited by existing vaccines. The secondcharacteristic of great public health concern is that influenza viruses,in particular influenza A virus can exchange genetic material and merge.This process, known as “antigenic shift”, results in new strainsdifferent from both parent viruses, which can be lethal pandemicstrains. Avian influenza is an influenza A virus that has crossed thespecies barrier from birds to mammals, including humans.

Avian Influenza

Most avian influenza (AI) strains are classified as low pathogenic avianinfluenza (LPAI) and cause few clinical signs in infected birds. Incontrast, high pathogenic avian influenza (HPAI) strains cause a severeand extremely contagious illness and death among infected birds.

Humans are not commonly affected by avian flu, however, the epidemics ofhighly pathogenic avian influenza (HPAI) recently seen in poultry inAsia increase opportunities for human exposure and infection. Severecases have been reported in the past and during the current epidemics inAsia. Recent highly pathogenic outbreaks have been caused by influenza Aviruses of subtypes H5 and H7. Of the 15 avian influenza virus subtypes,H5N1 is of particular concern since it mutates rapidly and has adocumented propensity to acquire genes from viruses infecting otheranimal species. Its ability to cause disease in humans is now welldocumented. Birds that survive infection excrete virus for at least 10days, orally and in feces, thus facilitating further spread at livepoultry markets and by migratory birds.

The epidemic of HPAI caused by H5N1, which began in mid-December 2003 incertain Asian countries has spread and poses a public health concern.The spread of infection in birds increases the opportunities for directinfection of humans. If more humans become infected over time, thelikelihood also increases that humans, if concurrently infected withhuman and avian influenza strains, could serve as the host for theemergence of a novel subtype with sufficient human influenza genes to beeasily transmitted from person to person. Such an event would mark thestart of an influenza pandemic.

AI spread between birds occurs primarily by direct contact betweenhealthy birds and infected birds, and through indirect contact withcontaminated equipment and materials. The virus is excreted through thefeces of infected birds and through secretions from the nose, mouth andeyes.

Contact with infected fecal material is the most common mode ofbird-to-bird transmission. Wild ducks often introduce low pathogenicityviruses into domestic flocks raised on range or in open flight pensthrough fecal contamination. Within a poultry house, transfer of theHPAI virus between birds can also occur via airborne secretions. Thespread of avian influenza between poultry premises almost always followsthe movement of contaminated people and equipment. Transfer of eggs isalso a potential means of AI transmission.

Influenza Virus Antigens and Vaccine Production

Immunization towards influenza virus is limited by the antigenicvariation of the virus and by the restriction of the infection to therespiratory mucous membranes. The influenza vaccines currently availableare based either on whole inactive virus, or on antigenic determinantsof the surface proteins. HA is a strong immunogen and is the mostsignificant antigen in defining the serological specificity of thedifferent virus strains.

The HA molecule (75-80 kD) comprises a plurality of antigenicdeterminants, several of which are in regions that undergo sequencechanges in different strains (strain-specific determinants) and othersin regions which are conserved in many HA molecules (commondeterminants). Due to these changes, flu vaccines need to be modified atleast every few years.

Many influenza antigens, and vaccines prepared therefrom, are known inthe art. U.S. Pat. No. 4,474,757 discloses a vaccine against influenzavirus infections consisting of a synthetic peptide corresponding to anantigenic fragment of HA attached to a suitable macromolecular carrier,such as polymers of amino acids or tetanus toxoid.

PCT International Publication WO 93/20846 to some of the inventors ofthe present invention teaches a synthetic recombinant vaccine against aplurality of different influenza virus strains comprising at least onerecombinant protein comprising the amino acid sequence of flagellin andat least one amino acid sequence of an epitope of influenza virus HA orNP, or an aggregate of said chimeric protein. Following this approach, asynthetic recombinant anti-influenza vaccine based on three epitopes wasfound to be highly efficient in mice. The exemplified vaccines includedflagellin chimeras comprising the HA 91-108 epitope, a B-cell epitopefrom the HA which is conserved in all H3 strains and elicitsanti-influenza neutralizing antibodies, together with one or bothT-helper or CTL NP epitopes (NP 55-69 and NP 147-158, respectively),which induce MHC-restricted immune responses. A vaccine comprising acombination of the three above mentioned chimeras was considered toafford the best protection to viral infection.

PCT application publication WO 00/32228 to some of the inventors of thepresent invention teaches a human synthetic peptide-based influenzavaccine comprising at least four epitopes of influenza virus, saidinfluenza virus epitopes being reactive with human cells, said epitopescomprising:

(i) one B-cell haemagglutinin (HA) epitope; (ii) one T-helperhaemagglutinin (HA) or nucleoprotein (NP) epitope that can bind to manyHLA molecules; and (iii) at least two cytotoxic lymphocyte (CTL)nucleoprotein (NP) or matrix protein (M) epitopes that are restricted tothe most prevalent HLA molecules in different human populations, inparticular specific ethnic or racial groups. The influenza peptideepitopes can be expressed as recombinant Salmonella flagellin. Thatvaccine requires the cumbersome preparation of at least four chimericpolypeptides.

PCT Application Publication WO 2004/080403 and US Patent ApplicationPublication US2004/0223976 provide a vaccine against disease caused byinfection with influenza virus, and methods of vaccination. Each vaccinecomprises a plurality of peptides derived from the M2 and/or HA proteinsof influenza virus chemically conjugated to a carrier protein. Theconjugation is between one terminus of the peptide and a reactive siteof the carrier protein where the carrier protein is selected from theouter membrane protein complex of Neisseria meningitidis, tetanustoxoid, hepatitis B surface antigen or core antigen, keyhole limpethemocyanin, rotavirus capsid protein, and the L1 protein of bovine orhuman papillomavirus VLP. That disclosure requires a plurality of M2 orHA peptide epitopes covalently bound to the outer surface of a carrierprotein and neither suggests nor teaches a vaccine comprising a chimericpolypeptide.

PCT Application Publication WO 99/07839 relates to influenza antigensfor use in vaccines wherein the vaccines are comprised of a fusionproduct of at least the extracellular part of M2 and a presentingcarrier. The M2 fragment was fused to the amino terminus of the carrierprotein in order to retain a free N-terminus of the M2-domain and inthis way mimic the wild type structure of the M2 protein. Furthermorethat invention is exemplified by way of a M2 fusion protein wherein theintact extracellular portion of M2 fragment is fused to the N-terminusof the hepatitis B virus core protein in order to mimic the wild typestructure of the M2 protein in viral particles and on infected cells,where the free N-terminus extends in the extracellular environment. Thatapplication neither teaches nor suggests an isolated M2 epitope that isconformationally constrained.

International Patent Application Publication No. WO 99/07839 teaches animmunogenic extracellular portion of a M2 membrane protein of aninfluenza A virus fused to a presenting carrier, which can be selectedfrom the amino terminus of the human Hepatitis B virus core protein,third complement protein fragment d (C3d), tetanus toxin fragment C oryeast Ty particles. Other non-peptidic presenting carriers arementioned, yet that invention is exemplified only by genetic fusionproducts.

Slepushkin et al. (1995) describes protection of mice to influenza Achallenge by vaccination with a recombinant M2 protein expressed inbaculovirus and administered with Freund's adjuvant.

PCT Application Publication No. WO 98/23735 discloses an influenzavaccine for inducing a cell-mediated cytolytic immune response againstan antigen in a mammal comprising a fusion product of an influenzaantigen and a stress protein or heat shock protein as carrier. Theinfluenza antigen is selected from hemagglutinin, nucleoprotein,neuraminidase, M1, M2, PB1, PB2, PA and a combination thereof. There isneither teaching nor suggestion of a vaccine combining a M epitope witha HA epitope.

Zou, et al. (2005) teach the extracellular M2 6-13 peptide and suggestthat that sequence may be useful in the preparation of an influenzavaccine. Liu, et al (2005) disclose host specific epitopes within theextracellular M2 sequences, which may be useful for the preparation of abivalent influenza vaccine. The relevant epitopes include the M2 10-20sequence common to human, avian and swine influenza.

PCT Application Publication No. WO 94/26903 relates to human influenzamatrix protein peptides able to bind to human MHC Class I molecules.That invention provides candidate peptide epitopes able to bind to thegroove of MHC class I molecules. identified using the antigen processingdefective cell line174.CEM T2 (T2). There is neither teaching norsuggestion of a vaccine comprising a combination of M peptide epitopewith a HA peptide epitope.

It would be highly advantageous to have an influenza vaccine that couldbe administered once and confer protection for several years or even alifetime by providing cross-protection against new strains of viruses.

There remains an unmet need for a vaccine useful in eliciting an immuneresponse to a broad range of influenza subtypes that affords long termand cross-species protection, is both cost effective and readilyproduced, can be administered in a variety of forms and is useful foranimal and human immunization.

SUMMARY OF THE INVENTION

The present invention provides an influenza vaccine eliciting long-termand cross-subtype protection against infection with influenza A andinfluenza B viruses, including the avian influenza serotypes. Anunexpectedly robust immune response to influenza A or B virus iselicited by a vaccine comprising chimeric proteins which comprise atleast one influenza A matrix protein (M) peptide epitope and at leastone influenza A or B haemagglutinin (HA) peptide epitope. A vaccinecomprising a combination of a M and a HA epitope overcomes drawbacks ofthe known vaccines including the need for including epitopes that arerestricted to HLA molecules associated with different racial or ethnicpopulations. The vaccine of the present invention elicits cross-racialefficacy and Asian and African populations react as well as Caucasians.

Additionally, a surprisingly effective immune response to influenza A orB virus is elicited by a vaccine comprising a chimeric proteincomprising a M2 peptide epitope and a flagellin amino acid sequencewherein the M2 peptide epitope is embedded within the flagellinpolypeptide sequence. This finding is unexpected in view of the hithertoknown M2 fusion proteins and conjugates that comprise at least theentire M2 extracellular region and mimic the conformation of the entireextracellular domain of the M2 protein.

In one aspect the present invention provides a vaccine for immunizationof a subject comprising a plurality of chimeric proteins comprising atleast two influenza virus peptide epitopes wherein the first peptideepitope is an influenza A virus matrix (M) peptide epitope and a secondpeptide epitope is a haemagglutinin (HA) peptide epitope, wherein thevaccine elicits cross strain protection.

In one embodiment the M peptide epitope is selected from a M1 or a M2peptide epitope. In various embodiments the M peptide epitope is derivedfrom the N-terminal domain of the M1 or M2 glycoprotein. The Mglycoprotein may be derived from any one of the influenza A virussubtypes, including H3N2, H5N1 and the like.

In some embodiments the M1 peptide epitope comprises from about 5 toabout 18 contiguous amino acids derived from the M1 N-terminal domain.In certain embodiments the M1 epitope comprises from about 8 to about 15contiguous amino acids derived from a MM1 N-terminal domain. Accordingto some embodiments the M1 epitope is selected from

M1 2-12 SLLTEVETYVP (SEQ ID NO: 26) M1 3-11 LLTEVETYV (SEQ ID NO: 27)M1 13-21 SIVPSGPL (SEQ ID NO: 28) M1 17-31 SGPLKAEIAQRLEDV(SEQ ID NO: 29) M1 18-29 GPLKAEIAQRLE (SEQ ID NO: 30)

In certain embodiments the M1 peptide epitope is selected from M1 2-12(SEQ ID NO:26) and M1 3-11 (SEQ ID NO:27).

In some embodiments the M2 peptide epitope comprises from about 5 toabout 20 contiguous amino acids derived from a M2 extracellular domain.In certain embodiments the M2 peptide epitope comprises from about 8 toabout 18 contiguous amino acids derived from a M2 extracellular domain.In certain embodiments M2 peptide epitope is conserved in all H3subtypes. In other embodiments the M2 peptide epitope is derived from aM2 extracellular domain of an H5, H7 or an H9 subtype.

In one embodiment the M2 peptide epitope comprises the M2 6-9 epitopehaving amino acid sequence EVET, set forth in SEQ ID NO:1. In someembodiments the M2 epitope is selected from the group consisting of

M2 3-11 peptide having amino acid sequence LLTEVETPI set forth in SEQ IDNO:6;

M2 2-10 peptide having amino acid sequence SLLTEVETP, set forth in SEQID NO:7;

M2 2-11 peptide having amino acid sequence SLLTEVETPI, set forth in SEQID NO:8;

M2 1-15 peptide having amino acid sequence MSLLTEVETHTRNGW set forth inSEQ ID NO:2.

M2 1-15 peptide having amino acid sequence MSLLTEVETPIRNEW, set forth inSEQ ID NO:10;

M2 1-18 peptide having amino acid sequence MSLLTEVETPIRNEWGCR, set forthin SEQ ID NO: 11;

M2 1-15 peptide having amino acid sequence MSLLTEVETLTKNGW set forth inSEQ ID NO:12;

M2 1-15 peptide having amino acid sequence MSLLTEVETLTRKGW set forth inSEQ ID NO:13; and

M2 6-13 peptide having amino acid sequence EVETPIRN, set forth in SEQ IDNO:20;

An exemplary list of M peptide epitopes useful in the vaccine of thepresent invention can be found in Table 1 herein below.

In various embodiments the HA epitope is an influenza A or influenza BB-cell type peptide epitope. In some embodiments the HA peptide epitopeis selected from the group consisting of HA 91-108 (SEQ ID NO:48), HA91-108 (SEQ ID NO:49), and HA 107-124 (SEQ ID NO:50). An exemplary listof HA epitopes useful in the vaccine of the present invention can befound in Table 2 herein below.

In some embodiments the HA peptide epitope is an influenza B HA peptideepitope. Exemplary influenza B HA peptide epitopes are HA 354-372 (SEQID NO:80) and 308-320 (SEQ ID NO:79).

According to some embodiments the vaccine of the present inventioncomprises the M1 2-12 (SEQ ID NO:26) and HA 91-108 (SEQ ID NO:48)influenza A peptide epitopes. In other embodiments the vaccine of thepresent invention comprises the M2 1-18 (SEQ ID NO:11) and HA 91-108(SEQ ID NO:48) influenza A peptide epitopes.

Additionally, the vaccine of the present invention may further compriseadditional antigenic peptides of influenza A or influenza B virus, inparticular B-cell type and T helper type peptide epitopes. In variousembodiments the vaccine further comprises at least one influenza A Thelper (Th) type peptide epitope. The T helper type peptide epitope maybe selected from a M, NP, HA or polymerase (PB) peptide epitope. Certainpreferred peptide epitopes include HA 307-319 (SEQ ID NO:57), HA 128-145(SEQ ID NO:56), HA 306-324 (SEQ ID NO:52), and NP 206-229 (SEQ ID NO:62)

In other embodiments the vaccine may further comprise one or more B-celltype peptide epitopes. Certain preferred B-cell type peptide epitopesinclude HA 150-159 (SEQ ID NO:51) and M2 6-13 (SEQ ID NO:20).

In various embodiments the vaccine may further comprise additionalinfluenza A or influenza B peptide epitopes. Certain preferred peptideepitopes include one or more of a B cell, Th or CTL type peptideepitopes or a combination thereof.

In some embodiments one or more CTL type epitopes that are restricted toprevalent HLA molecules in different populations are preferred. Examplesof peptide epitopes useful in the vaccine of the present invention maybe found in the tables set forth hereinbelow. Certain preferred CTL typepeptide epitopes include NP 335-350 (SEQ ID NO:66) or (SEQ ID NO:67), NP380-393 (SEQ ID NO:68).

In some embodiments the peptide vaccine comprises an influenza A virusM1 peptide epitope selected from M1 2-12 (SEQ ID NO:26) and M1 3-11 (SEQID NO:27); a HA 91-108 (SEQ ID NOS:48) peptide epitope and a Th typepeptide epitope selected from a Th HA peptide epitope and a Th NPpeptide epitope.

In other embodiments the peptide vaccine comprises an influenza A virusM2 peptide epitope selected from M2 1-18 (SEQ ID NO:11) and M2 1-15; aHA 91-108 peptide epitope and a Th type peptide epitope selected from aTh HA peptide epitope and a Th NP peptide epitope.

In some embodiments the vaccine comprises M1 1-12; HA 91-108; HA 307-319(SEQ ID NO:57): at least one CTL type NP peptide epitope selected fromthe group consisting of NP 335-350 (SEQ ID NO:67), NP 380-393 (SEQ IDNO:68), NP 265-273 (SEQ ID NO:63). In other embodiments the vaccinefurther comprises an influenza B peptide epitope. In some embodimentsthe influenza B epitope is HA 354-372 having amino acid sequence setforth in SEQ ID NO:80.

In one embodiment the vaccine comprises M1 1-12; HA 91-108; HA 307-319,NP 335-350, NP 380-393. In other embodiments the vaccine furthercomprises HA 354-372. In Other embodiments the synthetic vaccinecomprises M2 1-18; HA 91-108; HA 307-319, NP 335-350, NP 380-393. Inother embodiments the vaccine further comprises HA 354-372.

The peptide epitopes may be expressed as recombinant flagella, whereineach recombinant flagellin comprises one or more peptide epitopes.Alternatively, the peptide epitopes can be expressed in an expressionvector as a recombinant peptide protein which can be fused to aheterologous protein. In some embodiments an adjuvant or carrier isrequired. Accordingly, the present invention provides a synthetic orrecombinant peptide-based vaccine for immunization of a subjectcomprising at least two influenza virus epitopes wherein the firstepitope is an influenza A virus matrix (M) peptide epitope and a secondepitope is a haemagglutinin (HA) epitope; and a pharmaceuticallyacceptable adjuvant or carrier.

In another aspect the present invention provides a vaccine forimmunization of a subject comprising a plurality of chimeric proteinscomprising at least two influenza virus peptide epitopes wherein thefirst peptide epitope is an influenza A virus matrix (M) peptide epitopeand a second peptide epitope is a haemagglutinin (HA) peptide epitope;wherein each of said epitopes is expressed individually or together as achimeric polypeptide.

The chimeric polypeptide of the present invention does not includeconjugates in which peptide epitopes are conjugates to the outer surfaceof a carrier.

In some embodiments the present invention provides a vaccine comprisingat least two recombinant flagellin molecules wherein one recombinantflagellin comprises an influenza A virus M peptide epitope and a secondrecombinant flagellin comprises a HA peptide epitope.

The sequence of the flagellin is not limiting in the present inventionand can be selected from known flagellin sequences In certain preferredembodiments the recombinant flagellin comprises an amino sequence setforth in SEQ ID NO:161 (accession number CAA27130) and is encoded by apolynucleotide sequence set forth in SEQ ID NO:162. In other embodimentsthe chimeric polypeptide comprises modified flagellin.

The vaccine of the present invention is intended for human andveterinary applications including immunization of domestic animals (dog,cat, monkey etc.); livestock (horse, cow, sheep, goat, pig etc.), wildbirds (wild geese, wild ducks, etc.) and domestic birds (chicken, duck,geese etc.). Therefore, in addition to the M and HA epitopes the vaccineof the present invention may optionally further comprise Th and or CTLtype peptide epitopes selected from species specific strains ofinfluenza. The present invention further provides the use of at leastone influenza A M peptide epitope and at least one HA peptide epitopefor the preparation of a synthetic influenza vaccine.

In another aspect, the present invention provides a polynucleotideconstruct comprising a polynucleotide sequence of a nuclei acid sequenceof an influenza peptide epitope according to the present inventionoperably linked to an expression vector. The polynucleotide constructexpresses the chimeric polypeptide. The present invention furtherencompasses host cell comprising the polynucleotide construct.

In a third aspect the present invention provides a method for elicitingan immune response and conferring protection against influenza virus ina subject, wherein the method comprises administering to the subject avaccine comprising a plurality of chimeric proteins comprising at leasttwo influenza virus peptide epitopes wherein the first peptide epitopeis an influenza A virus matrix (M) peptide epitope and a second peptideepitope is a haemagglutinin (HA) peptide epitope, wherein the vaccineelicits cross strain protection.

In some embodiments the vaccine protects against influenza A, includingavian influenza. In other embodiments the vaccine comprises an influenzaB epitope and elicits protection against influenza B. In anotherembodiment the vaccine comprises peptide epitopes that elicit protectionto both influenza A and influenza B virus.

Routes of administration of the vaccine include, but are not limited tointraperitoneal, subcutaneous, intranasal, intramuscular, oral, topicaland transdermal delivery. Preferred routes of administration includeoral, intranasal (IN) and intramuscular (IM) administration. In oneembodiment the vaccine is formulated for intranasal administration. Inanother embodiment the vaccine is formulated for intramuscularadministration

It is to be explicitly understood that known compositions are to beexcluded from the present invention.

Further embodiments and the full scope of applicability of the presentinvention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a graph showing interferon gamma (IFNγ) secretion fromlymphocytes incubated with the respective peptide after the second andthird immunization of HHD/HLA A2 transgenic (TG) mice.

FIGS. 2A-2C show lysis of target cells by NK (Natural Killer cells)derived from mice immunized with individual or combination epitopesfollowing the second (2A) and third immunization (2B). Even after thesecond immunization, NK cells from the mice immunized with thecombination of 6 epitopes (hexa-vaccine1) were able to lyse the targetcells more efficiently than the cells from mice vaccinated with thenative flagella (2C). Specific lysis was determined at different E:Tratios.

FIGS. 3A and 3B show the results of immunizing C57Bl/6 mice withHexa-vaccine2, consisting of recombinant flagella comprising the HA91-108, HA 354-372, HA 307-319, NP 335-350, NP 380-393 and M2 1-18peptide epitopes. Serum was tested following 3 immunizations and thespecificity of antibody (Ab or Ig) against the whole H3N2 influenzavirus (3A) and against the specific M2 1-18 peptide (3B) was determined.

FIG. 4 shows binding of peptide epitopes to HLA-A2 on T2 cells: High anddose dependent binding was shown by the M1 3-11 peptide. Other testedpeptides, NP 336-344, NP 380-388 and HA 307-319, also showed bindingcapacity, which was not dose dependent.

FIGS. 5A-5C show antibody protection elicited by the recombinant peptideepitopes. A significant elevation in antibody (Ab) titer, specific toeach epitope was obtained with epitopes M1 2-12, NP 335-350 and HA91-108 in mice immunized with the single relevant epitope.

FIG. 6 shows the humoral immune dose response obtained followingvaccination of mice with Hexa-vaccine1.

FIG. 7 shows the humoral immune response to flagella followingintranasal and intramuscular administration of the Hexa-vaccine1.

FIG. 8 shows the results of virus titration. After three vaccinationswith Hexa-vaccine1, mice were infected with a sub-lethal dose ofinfluenza virus H3N2 strain (A/Texas/1/77). The lungs of the mice wereremoved 5 days later for titration of viral load. The titration wasperformed in fertilized eggs.

FIG. 9 presents the IgE titer in the dosing experiment and in theexperiment for evaluating the cellular response.

FIG. 10 shows IgE concentration (ng/ml) in sera of HHD transgenic miceimmunized intranasally (IN) with recombinant flagella expressinginfluenza epitopes.

FIG. 11 shows fold increase of IgG titer to 3 different influenzastrains in sera of NZW rabbits immunized intranasally three times withrecombinant flagella expressing six influenza epitopes (Hexa-vaccine-2).

FIG. 12 depicts pharmacokinetic data. Maximum serum concentration ofHexa-vaccine1 was observed after 15 minutes (T_(max)). Half (T_(1/2)) ofthe total exposure quantity was obtained within 30 minutes post dosing.Protein could not be detected after 12 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected discovery that avaccine comprising at least one M peptide epitope and at least one HApeptide epitope is able to elicit long-term and cross strain protectiveimmunity to influenza.

Definitions

For convenience, certain terms employed in the specification, examplesand claims are described herein.

The term “antigen presentation” means the expression of antigen on thesurface of a cell in association with major histocompatability complexclass I or class II molecules (MHC-I or MHC-II) of animals or with theHLA-I and HLA-II of humans.

The term “immunogenicity” or “immunogenic” relates to the ability of asubstance to stimulate or elicit an immune response. Immunogenicity ismeasured, for example, by determining the presence of antibodiesspecific for the substance. The presence of antibodies is detected bymethods known in the art, for example using an ELISA assay.

Influenza epitopes can be classified as B-cell type, T-cell type or bothB cell and T cell type, depending on the type of immune response theyelicit. The definition of B cell or T cell peptide epitope is notunequivocal; for example, a peptide epitope can induce antibodyproduction but at the same time that epitope can possess a sequence thatenables binding to the human HLA molecule, rendering it accessible toCTLs, hence a dual B cell and T cell classification for that particularepitope. “CTL”, “killer T cells” or “cytotoxic T cells” is a group ofdifferentiated T cells that recognize and lyse target cells bearing aspecific foreign antigen that function in defense against viralinfection and cancer cells. “T helper cell” or “Th” is any of the Tcells that when stimulated by a specific antigen release cytokines thatpromote the activation and function of B cells and killer T cells.

The term “recombinant flagellin” refers to a flagellin polypeptidecomprising a peptide epitope embedded within its sequence. A recombinantflagellin is distinct from a classical fusion protein in that thepeptide or protein being expressed in a fusion protein is fused to acarrier protein at either its N- or C-terminus, leaving the otherterminus free and conformationally unrestrained.

“Amino acid sequence”, as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules.

“Avian influenza” or “AI” refers to avian influenza virus that infectbirds, including domestic and wild birds. The known avian influenzaviruses belong to the H5, H7 and H9 virus subtypes. The avian influenzavirus may belong to the low pathogenic (LPAI) or high pathogenic type(HPAI) and may or may not have undergone antigenic shift. Certainstrains of avian flu, including H5N1, H7N3, H7N7 and H9N2, have beenshown to infect mammals, including humans.

Peptide Epitopes Useful in Preparing a Vaccine

Peptide epitopes derived from influenza proteins are useful in preparingthe composition of the present invention. A preferred compositionsincludes at least one peptide epitope derived from influenza A M1 or M2proteins in combination with an influenza A or influenza B HA peptideepitope. It is to be noted that peptide epitopes listed herein areprovided as for exemplary purposes only. The influenza virus proteinsvary between isolates, thereby providing multiple variant sequences foreach influenza protein. Accordingly, the present invention encompassespeptide epitopes having one or more amino acid substitutions, additionsor deletions.

The matrix protein M1 is a major structural component of the influenzavirus particles and forms an inner layer of the lipid cell-derivedenvelope. Within the virion and in infected cells at late stages of thevirus replication, the M1 protein associates with the viralribonucleoproteins (vRNPs), which are composed of viral RNA molecules,multiple copies of the NP, and the three subunits of the viralpolymerase holding the ends of the viral RNAs. The N-terminal domain ofM1 refers to amino acids 1 to about amino acid 20 of the M1 protein.

The matrix protein M2 is a hydrogen ion channel resulting indissociation of the matrix and nucleoprotein complex within vacuoles.This ion channel releases the genome enabling viral RNA to enter thenucleus of the infected cell and initiate viral replication. Therapeuticsubstances against influenza, such as amantadine and rimantadine act byblocking the M2 activity. Influenza B has a counterpart protein known asNB; although there is no sequence similarity they are both transmembraneproteins and may share similar function. The extracellular domain of theM2 protein which is a transmembrane protein of influenza A virus, isnearly invariant in all influenza A strains. The N-terminal domain of M2refers to the amino acid sequence N-terminal to the transmembranedomain.

Table 1 provides an exemplary list of M1 and M2 peptide epitopes thatmay be chosen for preparation of the chimeric proteins of the presentinvention.

TABLE 1 M1 and M2 peptide epitopes Epitope Epitope Amino Acid NucleotideType* Position Sequence Sequence NCBI # M2 6-9 EVET GAAGTGGAAACCABJ15715.1 (SEQ ID NO: 1) (SEQ ID NO: 81) Th M2 1-15 MSLLTEVETHTRNGATGAGCCTGCTGACC ABJ15715.1 W GAAGTGGAAACCCAC (SEQ ID NO: 2)ACCAGGAATGGGTGG (SEQ ID NO: 82) M2 10-18 PIRNEWGCR CCGATTCGTAACGAAABD59884 (SEQ ID NO: 3) TGGGGTTGTCGT (SEQ ID NO: 83) M2 8-15 ETPIRNEWGCGAAACCCCGATTCGT ABD59884 (SEQ ID NO: 4) AACGAATGGGGTTGT CGT(SEQ ID NO: 84) M2 10-20 PIRNEWGCRCN GAAACCCCGATTCGT ABD59884(SEQ ID NO: 5) AACGAATGGGGTTGT CGTGGTTGTCGT (SEQ ID NO: 85) CTL M2 3-11LLTEVETPI CTGCTGACCGAAGTGG ABD59884 (SEQ ID NO: 6) AAACCCCGATT(SEQ ID NO: 86) CTL M2 2-10 SLLTEVETP AGCCTGCTGACCGAAG ABD59884(SEQ ID NO: 7) TGGAAACCCCG (SEQ ID NO: 87) CTL M2 2-11 SLLTEVETPIAGCCTGCTGACCGAAG ABD59884 (SEQ ID NO: 8) TGGAAACCCCGATT (SEQ ID NO: 88)CTL M2 4-11 LTEVETPLT CTGACCGAAGTGGAAA ABD59884 (SEQ ID NO: 9)CCCCGCTGACC (SEQ ID NO: 89) Th M2 1-15 MSLLTEVETPIRNE ATGAGCCTGCTGACCGABD59884 W AAGTGGAAACCCCGAT (SEQ ID NO: 10) TCGCAACGAATGG(SEQ ID NO: 90) Th M2 1-18 MSLLTEVETPIRNE ATGAGCCTGCTGACCG ABD59884 WGCRAAGTGGAAACCCCGAT (SEQ ID NO: 11) TCGCAACGAATGGGGC TGCCGC (SEQ ID NO: 91)Th M2 1-15 MSLLTEVETLTKNG ATGAGCCTGCTGACCG AAK49250 W AAGTGGAAACCCTGAC(SEQ ID NO: 12) CAAAAACGGCTGG (SEQ ID NO: 92) Th M2 1-15 MSLLTEVETLTRNGATGAGCCTGCTGACCG ABI85097 W AAGTGGAAACCCTGAC (SEQ ID NO: 13)CCGCAACGGCTGG (SEQ ID NO: 93) CTL M2 4-12 LTEVETPIR CTGACCGAAGTGGAAAABD59884 (SEQ ID NO: 14) CCCCGATTCGC (SEQ ID NO: 94) CTL M2 4-13LTEVETPIRN CTGACCGAAGTGGAAA ABD59884 (SEQ ID NO: 15) CCCCGATTCGCAAC(SEQ ID NO: 95) CTL M2 6-14 EVETPIRNE GAAGTGGAAACCCCGA ABD59884(SEQ ID NO: 16) TTCGCAACGAA (SEQ ID NO: 96) CTL M2 6-15 EVETPIRNEWGAAGTGGAAACCCCGA ABD59884 (SEQ ID NO: 17) TTCGCAACGAATGG (SEQ ID NO: 97)CTL M2 4-14 LTEVETPIRNE CTGACCGAAGTGGAAA ABD59884 (SEQ ID NO: 18)CCCCGATTCGCAACGA A (SEQ ID NO: 98) Th M2 4-18 LTEVETPIRNEWGCCTGACCGAAGTGGAAA ABD59884 R CCCCGATTCGCAACGA (SEQ ID NO: 19)ATGGGGCTGCCGC (SEQ ID NO: 99) B cell M2 6-13 EVETPIRN GAAGTGGAAACCABD59900 (SEQ ID NO: 20) CCGATTCGTAAC (SEQ ID NO: 100) B cell M2 1-18MSLLTEVETPTRNE ATGAGCCTGCTGACCG BAD89348 WECR AAGTGGAAACCCCGAC(SEQ ID NO: 21) CCGCAACGAATGGGAA TGCCGC (SEQ ID NO: 101) B cell M2 2-24SLLTEVETPTRNEW AGCCTGCTGACCGAAG BAD89348 ECRCS DSSD TGGAAACCCCGACCCG(SEQ ID NO: 22) CAACGAATGGGAATGC CGCTGCAGCGATAGCA GCGAT (SEQ ID NO: 102)B cell M2 2-24 SLLTEVETPIRNEW AGCCTGCTGACCGAAG ABD59884 GCRCN DSSDTGGAAACCCCGATTCG (SEQ ID NO: 23) CAACGAATGGGGCTGC CGCTGCAACGATAGCA GCGAT(SEQ ID NO: 103) B cell M2 7-15 VETPIRNEW GTGGAAACCCCGATT ABD59884(SEQ ID NO: 24) CGTAACGAATGG (SEQ ID NO: 104) B cell M1 2-12 SLLTEVETYVLAGCCTGCTGACCGAAG AAO52904 (SEQ ID NO: 25) TGGAAACCTATGTGCT T(SEQ ID NO: 105) CTL M1 2-12 SLLTEVETYVP AGCCTGCTGACCGAAG AAO33507(SEQ ID NO: 26) TGGAAACCTATGTGCC G (SEQ ID NO: 106) CTL M1 3-11LLTEVETYV CTGCTGACCGAAGTGG AAO33507 (SEQ ID NO: 27) AAACCTATGTG(SEQ ID NO: 107) CTL M1 13-21 SIVPSGPL AGCATTGTGCCGAGCG ABD59901(SEQ ID NO: 28) GCCCGCTG (SEQ ID NO: 108) CTL M1 17-31 SGPLKAEIAQRLEDAGCGGCCCGCTGAAAG ABD59901 V CGGAAATTGCGCAGCG (SEQ ID NO: 29)CCTGGAAGATGTG (SEQ ID NO: 109) CTL M1 18-29 GPLKAEIAQRLEGGCCCGCTGAAAGCGG ABD59901 (SEQ ID NO: 30) AAATTGCGCAGCGCCT GGAA(SEQ ID NO: 110) CTL M1 27-35 RLEDVFAGK CGCCTGGAAGATGTGT ABD59901(SEQ ID NO: 31) TTGCGGGCAAA (SEQ ID NO: 111) CTL M1 41-51 ALMEWLKTRPIGCGCTGATGGAATGGC ABD59901 (SEQ ID NO: 32) TGAAAACCCGCCCG(SEQ ID NO: 112) CTL M1 50-59 PILSPLTKGI CCGATTCTGAGCCCGC ABD59901(SEQ ID NO: 33) TGACCAAAGGCATT (SEQ ID NO: 113) CTL M1 51-59 ILSPLTKGIATTCTGAGCCCGCTGA ABD59901 (SEQ ID NO: 34) CCAAAGGCATT (SEQ ID NO: 114)CTL M1 55-73 LTKGILGFVFTLTV CTGACCAAAGGCATTC ABD59901 PSERGTGGGCTTTGTGTTTAC (SEQ ID NO: 35) CCTGACCGTGCCGAGC GAACGCGGC(SEQ ID NO: 115) CTL M1 56-68 TKGILGFVFTLTV ACCAAAGGCATTCTGG ABD59901(SEQ ID NO: 36) GCTTTGTGTTTACCCT GACCGTG (SEQ ID NO: 116) CTL M1 57-68KGILGFVFTLTV AAAGGCATTCTGGGCT ABD59901 (SEQ ID NO: 37) TTGTGTTTACCCTGACCGTG (SEQ ID NO: 117) CTL M1 58-66 GILGFVFTL GGCATTCTGGGCTTTG ABD59901(SEQ ID NO: 38) TGTTTACCCTG (SEQ ID NO: 118) CTL M1 60-68 LGFVFTLTVCTGGGCTTTGTGTTTA ABD59901 (SEQ ID NO: 39) CCCTGACCGTG (SEQ ID NO: 119)CTL M1 59-67 ILGFVFTLT ATTCTGGGCTTTGTGT ABD59901 (SEQ ID NO: 40)TTACCCTGACC (SEQ ID NO: 120) CTL M1 128-135 ASCMGLIY GCGAGCTGCATGGGCCABD59901 (SEQ ID NO: 41) TGATTTAT (SEQ ID NO: 121) CTL M1 134-142RMGAVTTEV CGCATGGGCGCGGTGA ABD59901 (SEQ ID NO: 42) CCACCGAAGTG(SEQ ID NO: 122) CTL M1 145-155 GLVCATCEQIA GGCCTGGTGTGCGCGA ABD59901(SEQ ID NO: 43) CCTGCGAACAGATTGC G (SEQ ID NO: 123) CTL M1 164-172QMVATTNPL CAGATGGTGGCGACCA ABD59901 (SEQ ID NO: 44) CCAACCCGCTG(SEQ ID NO: 124) CTL M1 164-173 QMVATTNPLI CAGATGGTGGCGACCA ABD59901(SEQ ID NO: 45) CCAACCCGCTGATT (SEQ ID NO: 125) CTL M1 178-187RMVLASTTAK CGCATGGTGCTGGCGA ABD59901 (SEQ ID NO: 46) GCACCACCGCGAAA(SEQ ID NO: 126) CTL M1 232-240 DLLENLQTY GATCTGCTGGAAAACC ABD59901(SEQ ID NO: 47) TGCAGACCTAT (SEQ ID NO: 127)

Nucleoprotein (NP) is one of the groups of specific antigens, whichdistinguishes between influenza A, B and C viruses. In contrast to HA,NP is highly conserved, being 94% conserved in all influenza A viruses.Influenza A virus NP-specific antibody has no virus neutralizingactivity, but NP is an important target for cytotoxic T lymphocytes(CTL) which are cross-reactive with all type A viruses (Townsend, 1984).CTL recognize short synthetic peptides corresponding to linear regionsof the influenza NP molecule.

Hemagglutinin (HA) is a glycoprotein trimer embedded in the influenzaenvelope. It has responsible for the attachment and penetration of thevirus to the host cell. Antibodies to the HA neutralize viralinfectivity. Antigenic variations of this molecule are responsible forfrequent outbreaks of influenza and for the poor control of infection byimmunization (Ada and Jones, 1986).

The influenza virus RNA polymerase is a heterocomplex composed of thethree polymerase (P) proteins PB1, PB2 and PA-present in a 1:1:1 ratio.Their role in influenza virulence has not been fully elucidated.Non-limiting examples of HA, NP and PB peptide epitopes can be found intable 2 herein below.

TABLE 2 HA, NP and PB peptide epitopes. Epitope Epitope Amino AcidNucleotide NCBI Type* Position Sequence Sequence accession B cellHA 91-108 SKAYSNCYPYDVPD AGCAAAGCTTACAGCAAC AAM82562 YASL TGTTACCCTTAT(SEQ ID NO: 48) GATGTGCCGGATTAT GCCTCCCTT (SEQ ID NO: 128) B cellHA 91-108 SKAFSNCYPYDVPD AGCAAAGCGTTTAGCAAC CAC81017 YASLTGCTATCCGTATGATGTG (SEQ ID NO: 49) CCGGATTATGCGAGCCTG (SEQ ID NO: 129)B cell HA 107-124 STAYSNCYPYDVPD AGCACCGCGTATAGCAAC ABD59854 YASLTGCTATCCGTATGATGTG (from A/ (SEQ ID NO: 50) CCGGATTATGCGAGCCTG TW/3286/(SEQ ID NO: 130) 03 (H3N2) B cell HA 166-175 WLTEKEGSYP TGGCTGACGGAGAAGABD77675 (HA 150-159 (SEQ ID NO: 51) GAGGGCTCATACCCA A/PR/8(SEQ ID NO: 131) strain) Th HA 306-324 PKYVKQNTLKLATG CCCAAGTATGTTAAGCAAAAL62329 MRNVP AACACTCTGAAGTTGGCA (SEQ ID NO: 52) ACAGGGATGCGGAATGTACCAGAGAAACAAACTAGA GGC (SEQ ID NO: 132) CTL HA 521-531 GVKLESMGIYQGGCGTGAAACTGGAAAGC ABJ09518 (SEQ ID NO: 53) ATGGGCATTTATCAG(SEQ ID NO: 133) CTL HA 518-528 EISGVKLESNG GAAATTTCCGGCGTGAAA ABJ09518(SEQ ID NO: 54) CTGGAAAGCATGGGC (SEQ ID NO: 134) CTL HA 458-467NVKNLYEKVK AACGTGAAAAACCTGTAT ABD77675 (SEQ ID NO: 55) GAAAAAGTGAAA(SEQ ID NO: 135) Th HA 128-145 KVKILPKDRWTQHT AAAGTGAAAATTCTGCCGAAO46269 TTGG AAAGATCGCTGGACCCAG (SEQ ID NO: 56) CATACCACCACCGGCGGC(SEQ ID NO: 136) Th HA 307-319 PKYVKQNTLKLAT CCCAAGTATGTTAAGCAA AAL62329(HA 306-318) (SEQ ID NO: 57) AACACTCTGAAGTTGGCA ACA (SEQ ID NO: 137 ThNP 91-99 KTGGPIYRR AAAACTGGAGGACCT BAA99400 (SEQ ID NO: 58)ATATACAGGAGAGG (SEQ ID NO: 138) CTL NP 44-52 CTELKLSDYTGCACCGAACTGAAACTG BAA99400 (SEQ ID NO: 59) AGCGATTAT (SEQ ID NO: 139)CTL NP 82-95 HPSAGKDPKKTGGP CATCCGAGCGCGGGCAAA BAA99400 (SEQ ID NO: 60)GATCCGAAAAAAACCGGC GGCCCG (SEQ ID NO: 140) CTL NP 82-94 HPSAGKDPKKTGGCATCCGAGCGCGGGCAAA BAA99400 (SEQ ID NO: 61) GATCCGAAAAAAACCGGC GGC(SEQ ID NO: 141) Th NP 206-229 FWRGENGRKTRSAY TTTTGGCGCGGCGAAAACABD59868 ERMCNILKGK GGCCGCAAAACCCGCAGC (SEQ ID NO: 62)GCGTATGAACGCATGTGC AACATTCTGAAAGGCAAA (SEQ ID NO: 142) CTL NP 265-273ILRGSVAHK ATTCTGCGCGGCAGCGTG BAA99400 (SEQ ID NO: 63) GCGCATAAA(SEQ ID NO: 143) CTL NP 305-313 KLLQNSQVY AAACTGCTGCAGAACAGC ABD59868(SEQ ID NO: 64) CAGGTGTAT (SEQ ID NO: 144) CTL NP 335-349 SAAFEDLRVLSFIRAGCGCGGCGTTTGAAGAT ABD35694 G CTGCGCGTGCTGAGCTTT (SEQ ID NO: 65)ATTCGCGGC (SEQ ID NO: 145) CTL NP 335-350 SAAFEDLRVSSFIRAGCGCGGCGTTTGAAGAT ABK34765 GT CTGCGCGTGAGCAGCTTT (SEQ ID NO: 66)ATTCGCGGCACC (SEQ ID NO: 146) CTL NP 335-350 SAAFEDLRVLSFIRAGCGCGGCGTTTGAAGAT ABD35694 GY CTGCGCGTGCTGAGCTTT (SEQ ID NO: 67)ATTCGCGGCTAT (SEQ ID NO: 147) CTL NP 380-393 ELRSRYWAIRTRSGGAACTGCGCAGCCGCTAT ABK34765 (SEQ ID NO: 68) TGGGCGATTCGCACCCGC AGCGGC(SEQ ID NO: 148) CTL NP 380-388 ELRSRYWAI GAACTGCGCAGCCGCTAT ABK34765(SEQ ID NO: 69) TGGGCGATT (SEQ ID NO: 149) CTL NP 383-391 SRYWAIRTRAGCCGCTATTGGGCGATT BAA99400 (SEQ ID NO: 70) CGCACCCGC (SEQ ID NO: 150)CTL NP 384-394 YWAIRTRSGG TATTGGGCGATTCGCACC BAA99400 (SEQ ID NO: 71)CGCAGCGGCGGC (SEQ ID NO: 151 CTL NP 382-390 SRYWAIRTR AGCCGCTATTGGGCGATTBAA99400 (SEQ ID NO: 72) CGCACCCGC (SEQ ID NO: 152) CTL NP 418-426LPFDKPTIM CTGCCGTTTGATAAACCG BAA99400 (SEQ ID NO: 73) ACCATTATG(SEQ ID NO: 153) CTL PB1 591-599 VSDGGPNLY GTGAGCGATGGCGGCCCG ABK34974(SEQ ID NO: 74) AACCTGTAT (SEQ ID NO: 154) CTL PB1 571-579 RRSFELKKLCGCCGCAGCTTTGAACTG ABK34974 (SEQ ID NO: 75) AAAAAACTG (SEQ ID NO: 155)CTL PB2 368-376 RRATAILRK CGCCGCGCGACCGCGATT ABK34762 (SEQ ID NO: 76)CTGCGCAAA (SEQ ID NO: 156)

In some embodiments the vaccine further comprises a peptide epitopederived from influenza B. Non-limiting examples of influenza B peptideepitopes are shown in Table 3.

TABLE 3 Influenza B peptide epitopes Epitope Epitope Amino acidNucleotide NCBI type* position sequence sequence accession CTL NP 30-38RPIIRPATL CGCCCGATTATTCGCCC ABF21293 (flu B) (SEQ ID NO: 77) GGCGACCCTG(SEQ ID NO: 157) CTL NP 263-271 ADRGLLRDI GCAGATAGAGGGCTA ABF21293(flu B) (SEQ ID NO: 78) TTGAGAGACATC (SEQ ID NO: 158) Th HA 308-320PYYTGEHAKAIGN CCGTATTATACCGGCGA ABI84095 (flu B) (SEQ ID NO: 79)ACATGCGAAAGCGATTG GCAAC (SEQ ID NO: 159) B HA 354-372 PAKLLKERGFFGAICCGGCGAAACTGCTGAA ABI83926 (flu B) AGFLE AGAACGCGGCTTTTTTG(SEQ ID NO: 80) GCGCGATTGCGGGCTTT CTGGAA (SEQ ID NO: 160) *Each peptidemay belong to one or more epitope types. For example, a peptide thatelicits a B-cell response can also elicit a T-cell (Th and/or CTL)response.Nucleic Acids

The present invention further provides nucleic acid molecules encodingthe a vector such as an expression vector comprising the influenzaepitopes and a host cell comprising a vector which comprises aninfluenza epitope useful in the preparation of a synthetic vaccine ofthe invention.

An isolated nucleic acid sequence encoding a peptide can be obtainedfrom its natural source, for example as a portion of a gene. A nucleicacid molecule can also be produced using recombinant DNA technology(e.g., polymerase chain reaction (PCR) amplification, cloning) orchemical synthesis. Nucleic acid sequences include natural nucleic acidsequences and homologs thereof, comprising, but not limited to, naturalallelic variants and modified nucleic acid sequences in whichnucleotides have been inserted, deleted, substituted, and/or inverted insuch a manner that such modifications do not substantially interferewith the nucleic acid molecule's ability to encode a functionalflagellin of the present invention.

A polynucleotide or oligonucleotide sequence can be deduced from thegenetic code of a protein, however, the degeneracy of the code must betaken into account, and nucleic acid sequences of the invention alsoinclude sequences, which are degenerate as a result of the genetic code,which sequences may be readily determined by those of ordinary skill inthe art.

The terms “nucleic acid” and “polynucleotide” as used herein refer to anoligonucleotide, polynucleotide or nucleotide and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin, which may besingle- or double-stranded, and represent the sense or antisense strand.The term should also be understood to include, as equivalents, analogsof either RNA or DNA made from nucleotide analogs, and, as applicable tothe embodiment being described.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms“amplimers”, “primers”, “oligomers”, and “probes”, as commonly definedin the art. The oligonucleotides encoding the specific peptide epitopesuseful in the preparation of the vaccine of the present invention areprovided in tables 1-3 hereinabove.

As used herein, highly stringent conditions are those, which aretolerant of up to about 5% to about 25% sequence divergence, preferablyup to about 5% to about 15%. Without limitation, examples of highlystringent (−10° C. below the calculated Tm of the hybrid) conditions usea wash solution of 0.1×SSC (standard saline citrate) and 0.5% SDS at theappropriate Ti (incubation temperature) below the calculated Tm of thehybrid. The ultimate stringency of the conditions is primarily due tothe washing conditions, particularly if the hybridization conditionsused are those, which allow less stable hybrids to form along withstable hybrids. The wash conditions at higher stringency then remove theless stable hybrids. A common hybridization condition that can be usedwith the highly stringent to moderately stringent wash conditionsdescribed above is hybridization in a solution of 6×SSC (or 6×SSPE),5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured, fragmented salmonsperm DNA at an appropriate Ti. (See generally Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring HarborPress (1989) for suitable high stringency conditions).

Stringency conditions are a function of the temperature used in thehybridization experiment and washes, the molarity of the monovalentcations in the hybridization solution and in the wash solution(s) andthe percentage of formamide in the hybridization solution. In general,sensitivity by hybridization with a probe is affected by the amount andspecific activity of the probe, the amount of the target nucleic acid,the detectability of the label, the rate of hybridization, and theduration of the hybridization. The hybridization rate is maximized at aTi of about 20° C.-25° C. below Tm for DNA:DNA hybrids and about 10°C.-15° C. below Tm for DNA:RNA hybrids. It is also maximized by an ionicstrength of about 1.5M Na⁺. The rate is directly proportional to duplexlength and inversely proportional to the degree of mismatching.

Specificity in hybridization, however, is a function of the differencein stability between the desired hybrid and “background” hybrids. Hybridstability is a function of duplex length, base composition, ionicstrength, mismatching, and destabilizing agents (if any). The Tm of aperfect hybrid may be estimated for DNA:DNA hybrids using the equationof Meinkoth et al (1984).

Chimeric or Recombinant Molecules

A “chimeric protein”, “chimeric polypeptide” or “recombinant protein”are used interchangeably and refer to an influenza peptide epitopeoperatively linked to a polypeptide other than the polypeptide fromwhich the peptide epitope was derived. The peptide epitopes of thepresent invention can be prepared by expression in an expression vectoras a chimeric protein. The methods to produce a chimeric or recombinantprotein comprising an influenza peptide epitope are known to those withskill in the art. A nucleic acid sequence encoding an influenza peptideepitope can be inserted into an expression vector for preparation of apolynucleotide construct for propagation and expression in host cells.

In a non-limiting example, the chimeric polypeptide of the presentinvention includes chimeras of an influenza peptide epitope with one ofthe following polypeptides: flagellin, Cholera toxin, Tetanus toxin,Ovalbumin, Tuberculosis heat shock protein, Diphtheria Toxoid, Protein Gfrom respiratory syncytial virus, Outer Membrane Protein from Neisseriameningitides, nucleoprotein (N) of vesicular stomatitis virus,glycoprotein (G) of vesicular stomatitis virus, Plasmodium falciparumAntigen Glutamate-Rich Protein, Merozoite Surface Protein 3 or Virusesenvelope (E) protein.

The term “expression vector” and “recombinant expression vector” as usedherein refers to a DNA molecule, for example a plasmid, flagellin orvirus, containing a desired and appropriate nucleic acid sequencesnecessary for the expression of the recombinant peptide epitopes forexpression in a particular host cell. As used herein “operably linked”refers to a functional linkage of at least two sequences. Operablylinked includes linkage between a promoter and a second sequence, forexample an nucleic acid of the present invention, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence.

The regulatory regions necessary for transcription of the peptideepitope can be provided by the expression vector. The precise nature ofthe regulatory regions needed for gene expression may vary among vectorsand host cells. Generally, a promoter is required which is capable ofbinding RNA polymerase and promoting the transcription of anoperably-associated nucleic acid sequence. Regulatory regions mayinclude those 5′ non-coding sequences involved with initiation oftranscription and translation, such as the TATA box, capping sequence,CAAT sequence, and the like. The non-coding region 3′ to the codingsequence may contain transcriptional termination regulatory sequences,such as terminators and polyadenylation sites. A translation initiationcodon (ATG) may also be provided.

In order to clone the nucleic acid sequences into the cloning site of avector, linkers or adapters providing the appropriate compatiblerestriction sites during synthesis of the nucleic acids. For example, adesired restriction enzyme site can be introduced into a fragment of DNAby amplification of the DNA by use of PCR with primers containing thedesired restriction enzyme site.

An expression construct comprising a peptide epitope sequence operablyassociated with regulatory regions can be directly introduced intoappropriate host cells for expression and production of the peptideepitopes per se or as recombinant fusion proteins. The expressionvectors that may be used include but are not limited to plasmids,cosmids, phage, phagemids, flagellin or modified viruses. Typically,such expression vectors comprise a functional origin of replication forpropagation of the vector in an appropriate host cell, one or morerestriction endonuclease sites for insertion of the desired genesequence, and one or more selection markers.

The recombinant polynucleotide construct comprising the expressionvector and a peptide epitope should then be transferred into a bacterialhost cell where it can replicate and be expressed. This can beaccomplished by methods known in the art. The expression vector is usedwith a compatible prokaryotic or eukaryotic host cell which may bederived from bacteria, yeast, insects, mammals and humans.

A particularly preferred expression vector is a flagellin vector. Anon-limiting example of a flagellin expression vector is disclosed inU.S. Pat. No. 6,130,082 incorporated herein by reference. Otherexpression vectors which include a flagella gene, for example asalmonella fliC gene are also suitable. The host cells which express therecombinant flagellin can be formulated as live vaccines.

An alternative to producing the peptide epitopes by recombinanttechniques is peptide synthesis by use of a peptide synthesizer.Conventional peptide synthesis or other synthetic protocols well knownin the art may be used.

Vaccine Formulation

The vaccine can be formulated for administration in one of manydifferent modes. According to one embodiment of the invention, thevaccine is administered intranasally. The intranasal composition can beformulated, for example, in liquid form as nose drops, spray, orsuitable for inhalation, as powder, as cream, or as emulsion. Thecomposition can contain a variety of additives, such as adjuvant,excipient, stabilizers, buffers, or preservatives. For straightforwardapplication, the vaccine composition is preferably supplied in a vesselappropriate for distribution of the recombinant flagellin in the form ofnose drops or an aerosol. In certain preferred embodiments the vaccineis formulated for mucosal deliver, in particular nasal delivery (Arnon,2001; Ben-Yedidia, 1999).

In another embodiment of the invention, administration is oral and thevaccine may be presented, for example, in the form of a tablet orencased in a gelatin capsule or a microcapsule.

In yet another embodiment, the vaccine is formulated for parenteraladministration. In some embodiments the vaccine is formulated for massinoculation, for example for use with a jet-injector or a single usecartridge.

The formulation of these modalities is general knowledge to those withskill in the art.

The liposome provides another delivery system for antigen delivery andpresentation. Liposomes are bilayered vesicles composed of phospholipidsand other sterols surrounding a typically aqueous center where antigensor other products can be encapsulated. The liposome structure is highlyversatile with many types range in nanometer to micrometer sizes, fromabout 25 nm to about 500 μm. Liposomes have been found to be effectivein delivering therapeutic agents to dermal and mucosal surfaces.Liposomes can be further modified for targeted delivery by for example,incorporating specific antibodies into the surface membrane, or alteredto encapsulate bacteria, viruses or parasites. The average survival timeof the intact liposome structure can be extended with the inclusion ofcertain polymers, for example polyethylene glycol, allowing forprolonged release in vivo.

Microparticles and nanoparticles employ small biodegradable sphereswhich act as depots for vaccine delivery. The major advantage thatpolymer microspheres possess over other depot-effecting adjuvants isthat they are extremely safe and have been approved by the Food and DrugAdministration in the US for use in human medicine as suitable suturesand for use as a biodegradable drug delivery system (Langer, 1990). Therates of copolymer hydrolysis are very well characterized, which in turnallows for the manufacture of microparticles with sustained antigenrelease over prolonged periods of time (O'Hagen, et al., 1993).

Parenteral administration of microparticles elicits long-lastingimmunity, especially if they incorporate prolonged releasecharacteristics. The rate of release can be modulated by the mixture ofpolymers and their relative molecular weights, which will hydrolyze overvarying periods of time. Without wishing to be bound to theory, theformulation of different sized particles (1 μm to 200 μm) may alsocontribute to long-lasting immunological responses since large particlesmust be broken down into smaller particles before being available formacrophage uptake. In this manner a single-injection vaccine could bedeveloped by integrating various particle sizes, thereby prolongingantigen presentation and greatly benefiting livestock producers.

In some applications an adjuvant or excipient may be included in thevaccine formulation. The choice of the adjuvant will be determined inpart by the mode of administration of the vaccine. For example,non-injected vaccination will lead to better overall compliance andlower overall costs. A preferred mode of administration is intranasaladministration. Non-limiting examples of intranasal adjuvants includechitosan powder, PLA and PLG microspheres, QS-21, calcium phosphatenanoparticles (CAP) and mCTA/LTB (mutant cholera toxin E112K withpentameric B subunit of heat labile enterotoxin).

Therapeutic Use of Vaccine

The vaccines of the invention are intended both for use in humans and inanimals including livestock, poultry and domestic animals, forprevention or attenuation of influenza A and B disease.

The present invention provides a method for inducing an immune responseagainst influenza virus. The method comprises administering to asubject, which is an animal or a human subject, a vaccine comprisingrecombinant flagellin according to the principles of the presentinvention.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES

ABBREVIATIONS: Ab: Antibodies; CTL: Cytotoxic T-lymphocytes; EID:Egg-infective dose; HA: Hemagglutinin; HAU: Hemagglutination unit; NP:Nucleoprotein; PMBC: Peripheral blood mononuclear cells; Th: T helper;IN or i.n.: intranasal; IP: intraperitoneal; IM or i.m.: intramuscular;NK: natural killer cells; E:T ratio: effector target ratio.

Example 1 Synthesis of Peptides and Recombinant Flagellin

The epitopes may be expressed as recombinant flagella, wherein eachflagella comprises one or more peptide epitopes. Alternatively, thepeptide epitopes can be expressed in an expression vector as amultimeric fusion protein comprising two or more peptide epitopes.

The recombinant flagellin are synthesized by molecular biology methodsknown in the art. In brief: a flagella expression vector comprising anantibiotic marker was prepared. The synthesized oligonucleotides wereinserted at the EcoRV site of the plasmid and transfected into E. colicompetent cells. Colonies containing the recombinant plasmid wereselected by probing with a radiolabeled oligonucleotide. Plasmids frompositive colonies were purified and the insert orientation wasdetermined using restriction analysis. The desired plasmids were used totransform Salmonella typhimurium LB5000 (a restrictive negative,modification proficient non flagellated) competent cells and were thentransferred to a flagellin negative live vaccine strain (an Aro Amutant) of Salmonella dublin SL5928 by transduction using the phageP22HT105/1 int. The transformed S. dublin were selected for kanamycinresistance, motility under the light microscope and growth in semisolidLB agar plates, supplemented with Oxoid nutrient broth no. 2. Selectedclones were grown overnight in 2 liters of medium and the flagellinpurified by acidic cleavage. The resulting product is an aggregate ofthe flagellin and may be used as such for a vaccine.

The recombinant flagellin is not intended to be limited by the choice ofthe flagellin sequence. In some embodiments the flagellin havingaccession number X03395 Salmonella muenchen H1-d gene for phase-1-dflagellin (ATCC 8388).

Example 2 Response of Chimeric Mice to Whole Inactivated Influenza Virus

Human mouse chimeras are used to evaluate the immunogenic responseelicited by the vaccine of the present invention. In order to establishthe suitability of the human/mouse radiation chimera for evaluating thepeptide-based vaccine, their immune response to inactive purifiedinfluenza virus was evaluated. The mice were immunized i. p. with 50 μgof the virus on the day of PBMC transplantation, followed by a sublethalviral challenge with influenza A/Texas/1/77 strain 14 days afterimmunization. The vaccination of human/mouse radiation chimera with thewhole killed virus vaccine, without any adjuvant, induced production ofspecific antibodies—the serum antibody titer was significantly higher(2.4 fold) in the immunized chimera as compared to the control group.Moreover, this vaccination markedly reduced the subsequent virusinfection. The lung virus titer after challenge was significantly lower(by 2.7 orders of magnitude) in the immunized chimera as compared to thecontrol group.

This experiment demonstrates the suitability of the human/mouseradiation chimera for evaluating the anti-influenza response followingimmunization.

Example 3 FACS Analysis of Immunized Mice for Evaluating the Engraftmentof Human PBMC in Human/BALB Chimera

The successful engraftment of the human cells in the human/mousechimeras demonstrated in a preliminary experiment showing that most ofthe lymphocytes in the peritoneum (50-80%) and in the lungs of the mice(30-60%) were of human origin. For the evaluation of human cellengraftment in the human/mouse chimera, the presence of human cells inthe engrafted mice was analyzed by FACS.

Example 4 Virus Clearance from the Lungs Following Sub-Lethal Challenge

Influenza infection is a respiratory disease; hence, a local immuneresponse induced by an intranasal administration of the vaccine may bemore efficient than parenteral administration. The immunization schedulewas modified in order to adapt it for intranasal immunization.

Mice (6-8 per group in 7 repeated experiments) are immunizedintranasally (i. n.) 10-12 days after PBMC transplantation, as describedin the Methods. Ten days later, they are challenged i. n. with 10-4 HAUin 50 μl allantoic fluid of live A/Texas/1/77 strain or another strainof influenza virus. Five days later they were sacrificed and their lungswere removed for virus titration. Human antibody production in thesemice is evaluated in both the serum (before challenge) and in the lungs(after challenge).

Further to the sub-lethal infection challenge experiment, the ability ofthe vaccine to protect human/mouse chimera from a lethal dose ofinfluenza virus is examined.

Example 5 Protection from Infection with Different Strains of Influenza

One of the major problems with currently available influenza vaccines isthat they are effective only against the strains included in thevaccine. Therefore, it is of interest to examine the ability of therecombinant flagellin comprising influenza epitopes to protect mice fromdifferent influenza subtypes. In one embodiment the M peptide epitope,which is expressed in the flagellin, is conserved in all influenza H3subtypes, while the T-cell epitopes are from regions of thehaemagglutinin and nucleoprotein highly conserved in other subtypes aswell. In other examples the M peptide epitope is conserved in all H9 orH5 strains. In the first step, it is shown that rabbit antibodiestowards these epitopes can indeed recognize and react in ELISA withdifferent strains of influenza including A/Texas/1/77, A/Aichi/68,A/PR/8/34 and A/Japanese/57. To further test the potential of theseepitopes to confer cross protection in humans, the human/mouse radiationchimera (8 mice per group) were immunized i. n. with the recombinantflagellin. Their resistance to different influenza strains challenge wasdetected 7 days later and compared to non-transplanted mice that areimmunized with the same flagella mixture. The influenza strains used forinfection were: A/Texas/1/77 (H3N2), A/Japanese/57 (H2N2) and A/PR/8/34(H1N1). Other strains are tested, as well.

Example 6 CTL Response Assay

Following immunization with the vaccine of the present invention, theanimals' spleens are removed and incubated with the peptide representingthe relevant epitope (for example NP 335-350 or M2 1-18) for anadditional 5 days. TAP deficient syngeneic target cells (with the sameHLA A2.1 typing, for example: RMA cells) are incubated with the samepeptide and with S³⁵-Methionine in a separate dish. The activatedsplenocytes are incubated with the target cell and upon recognition ofthe peptide, will attack them and release of methionine. Highradioactivity counts indicate a high level of active CTL cells.

Example 7 Serum Antibody and Antibody Neutralization Assays

Mice, guinea pigs or rabbits are immunized with the mixture ofrecombinant flagellin, 14 days post immunization, blood samples aretaken and serum is separated. These sera can be employed for detectionthe specificity of the antibodies, for example by ELISA or western blot,in addition, it is possible to determine their type, i.e. IgG1, or IgG2aor IgG2c etc. In lung homogenates IgA can be detected, indicative of amucosal immune response.

Neutralization assay: MDCK cells are infected by influenza virus asdetermined by plaque counts or by MTT (sort of viability staining),following incubation of the virus with serum from immunized animals; theantibodies bind the virus and prevent infection of the MDCK cells. Areduced number of plaques or increased viability of the cells indicatethe specificity of the antibodies and their ability to prevent/reduceinfection.

Example 8 Immunogenicity of Individual Epitopes

HHD/HLA A2 transgenic mice were immunized with flagella expressing eachepitope (Fla-91, Fla-335, Fla-380, Fla-M1 2-12) or with a mixture of 6epitopes (Hexa-vaccine1: Fla-91, Fla-335, Fla-380, Fla-M1 2-12, Fla-354and Fla-307). Fla-91 refers to flagella comprising HA 91-108 epitope;Fla-335 refers to flagella comprising NP 335-350 epitope; Fla-380 refersto flagella comprising NP 380-393 epitope; Fla-M1 2-12 refers toflagella expressing M1 2-12 epitope; Fla-354 refers to flagellaexpressing HA 354-372 (influenza B) epitope; Fla-307 refers to flagellaexpressing HA 307-319 epitope.

The cells were incubated with the respective peptide and the cells fromHexa-vaccine immunized mice were incubate with the 4 peptides HA91,NP335, NP380, M1 2-12. The peptide epitope sequences, sequenceidentifiers and strain homology data can be founds in table 4 hereinbelow.

TABLE 4 Hexa vaccine1 peptide epitope list Epitope Homology in typeEpitope sequence Influenza strains Th HA 307-319 H3N2 PKYVKQNTLKLAT(SEQ ID NO: 57) CTL NP 335-350 H1N2, H2N2, H3N2, SAAFEDLRVLSFIRGY H9N2.(SEQ ID NO: 67) CTL NP 380-393 H1N1, H1N2, H2N2, ELRSRYWAIRTRSGH3N8, H5N1, H5N2, (SEQ ID NO: 68) H5N9, H6N1, H6N2, H6N9, H7N7, H9N2,H9N2, H11N1, H11N8, H11N9, H14N5. B-cell HA 91-108 H3N2SKAYSNCYPYDVPDYASL (SEQ ID NO: 48) B-cell M1 2-12 H1N1, H3N2, H4N6,and CTL SLLTEVETYVP H5N1, H5N2, H5N3, (SEQ ID NO: 26) H6N1, H7N3, H9N2,B-cell HA 354-372 B/HongKong/330/2001; (Influ- PAKLLKERGFFGAIAGFLEB/Beijing/1/87; enza B) (SEQ ID NO: 80) B/Singapore/222/79;B/Oregon/5/80; B/Shangdong/7/97; B/Memphis/13/03; B/Los Angeles/1/02;B/Nebraska/1/01; B/Hong Kong/548/2000; B/Hong Kong/156/99;B/Vienna/1/99; B/Lee/40 and othersImmunogenicity in “HHD Transgenic Mouse” Model

The CTL epitopes were selected for their binding to HLA-A2 molecules. Inorder to study HLA-restricted responses, the D b−/− β2 microglobulin(β2m) null mice, transgenic for a recombinant HLA-A2.1/D b−/−. β2microglobulin single chain (HHD mice) was employed. These mice combineclassical HLA transgenesis with selective destruction of murine H-2 andshow only HLA-A2.1-restricted responses. These mice serve as an animalmodel for research of systems involving cellular immunity such ascancer, autoimmunity and vaccination issues.

The cellular response that contributes to the elimination of the virusinvolves cytokine-mediated mechanisms. The involvement of cytokines inthe immune response mounted by the recombinant vaccine was studied inthe HHD transgenic mice model.

In this study, flagella expressing various peptide epitopes in PBS,emulsified in Freund adjuvant, were administered subcutaneously threetimes. The control group was administered with PBS emulsified in Freundadjuvant.

Cellular assays: splenocytes of these mice were incubated in thepresence of the synthetic peptides corresponding to the above epitope.IFN-γ secretion by the cells in response to the stimulation with thepeptides was monitored by ELISA.

FIG. 1 shows the IFN gamma secretion as measured from lymphocytesincubated with the respective peptide after the second and thirdimmunization.

Activated lymphocytes secreted IFN-γ in response to incubation with thecorresponding peptides. The group immunized with Hexa-vaccine1,containing the mixture of 6 recombinant flagella was incubated with amixture of the 4 cellular epitopes tested separately. After the thirdimmunization, The IFN-γ secretion from these cells is significantlyelevated (IFN levels secreted by cells incubated with medium, were belowthe assay detection level of 2 pg/ml). The IFN gamma secreted from nonactivated lymphocytes (negative controls—grown in medium withoutpeptide) was <0.004 ng/ml.

NK (Natural Killer cell) lysis contributes to the anti viral response.It is known that viral infected cells are more sensitive to lysis thannon-infected cells. It is speculated that the recognition of targetcells by NK cells is more ‘specific’ than previously thought. Asimilarity in peptide motif between HLA A2 binders and HLA-G (expressedon NK cells) binders has been demonstrated.

Therefore, in addition to the non-specific mechanism of NK activation,peptides specific to HLA-A2 can elicit further specific elevation of NKactivity (lysis).

Cytotoxic T lymphocyte (CTL) assays: HHD/HLA A2.1 mice were immunizedwith Hexa-vaccine1 in PBS. Direct lysis of target cells by CD8+lymphocytes was not demonstrated, however, a marked lysis of Yac-1 cellsthat are sensitive to NK lysis was obtained. Lysis by NK was found afterthe second immunization and was further elevated after the thirdimmunization. It should be noted that baseline levels of NK activationin naïve mice is approximately null.

FIGS. 2A-2C shows lysis of target cells by NK derived from vaccinatedmice was followed after the second and third immunization. Percentage oflysis of YAC-1 targets by NK cells after the second and thirdimmunization is presented. Splenic lymphocytes from immunized mice weresensitized with the peptides included in the recombinant flagella for 5days and then incubated with ³⁵S-Met labeled YAC-1 cells. Specific lysiswas determined at different E:T ratios. The data (% lysis by NK cells ofeach group) is presented as fold activation in comparison to the lysisof the group immunized with native flagella. After the secondimmunization, NK cells from the mice immunized with the combination of 6epitopes (Hexa-vaccine1) were able to lyse the target cells moreefficiently than the cells from mice vaccinated with the nativeflagella. FIGS. 2A and 2B show the NK cell lysis of individualrecombinant peptide epitopes or a combination of three recombinantepitopes (Fla-NP335, Fla-NP380, Fla-M1 2-12).

After three immunizations, the cells from mice immunized with M1 2-12showed a significant elevation in their ability to destroy target cells.The M1 2-12 peptide epitope is therefore a useful peptide epitope in thepreparation of the vaccine of the present invention.

FIGS. 3A and 3B show the results of immunization of C57Bl/6 mice withthe Hexa-vaccine1, consisting of flagella with 6 influenza epitopes (HA91-108, HA 354-372, HA 307-319, NP 335-350, NP 380-393 and M2 1-18):

Serum was removed after a schedule of 3 immunizations and thespecificity of Ab against the whole H3N2 influenza virus (FIG. 3A) andagainst the specific M2 1-18 peptide (FIG. 3B) was determined.

Binding of epitopes and stabilization of HLA-A2 on human T2 (deficientfor TAP transporters and therefore express low and unstable amounts ofHLA-A2.1 molecules. Upon binding of peptides, stable and high levels ofHLA-A2 are expressed on the cell surface). T2 cells were incubated withvarious concentrations of peptides in serum-free medium over night andstained with specific monoclonal antibodies. Stabilization of HLA-A2molecules was detected by Flow cytometry.

FIG. 4 shows binding of peptides to HLA-A2 on T2 cells: High and dosedependent binding was shown by the M1 3-11 peptide. Other testedpeptides, NP 336-344, NP 380-388 and HA 307-319, showed some low bindingcapacity, which was not dose dependent. NP 380-393 is known as specificto HLA B8 molecule and is not expected to bind HLA A2. The HA 91-108peptide which is longer than the HLA groove, showed binding capacityonly at the higher (100 μM) concentration, probably due to the HLA motifin its N-terminal side. Minimal and not dose dependent binding wasdemonstrated at lower concentrations.

In addition to the cellular responses, sera from mice after the thirdimmunization were compared to pre immune sera for antibodies specific tothe epitopes:

FIGS. 5A-5C shows that the recombinant peptide epitopes elicit antibodyprotection. A significant elevation in Ab titer, specific to the epitopewas obtained with epitopes NP 335-350 (5A), M1 2-12 (5B) and HA 91-108(5C) in mice immunized with the single relevant epitope (200 μg/mouse,IM).

Conclusions

The results of this study indicate that immunization with therecombinant flagellin containing specific T cells epitopes resulted inspecific recognition and cellular TH1 type response, as shown by IFN-γsecretion by lymphocytes from the vaccinated mice in response toin-vitro stimulation with the respective synthetic peptides.Furthermore, the specific binding of the investigated epitopes to HLA A2expressing target cells as well as specific lysis of these cells loadedwith the epitope by NK cells indicates cellular response to theHexa-vaccine1 in HHD transgenic mice.

Example 9 Dose Optimization Study

This study was designed to select the optimal dose of the epitope basedanti influenza vaccine Hexa-vaccine, which is the lowest effective doseconferring sufficient immune response as measured by variousimmunological tests. Performing this study with Hexa-vaccine assessedthe efficacy of the vaccine by assessment of humoral response and virustitration in the lungs after a schedule of 3 immunizations andinfection.

Study Design

HHD/HLA A2.1 mice were immunized 3 times IN and IM with 240, 80, 24 or 8μg of Hexa-vaccine1, consisting of influenza epitopes within bacterialflagella in PBS (see Table 5 for groups and identification). Blood wascollected for evaluation of the humoral response elicited in thevaccinated mice. The mice were infected with H3N2 influenza virus andviral titration in their lungs served as a correlate for efficacy.

TABLE 5 Study Groups and Identifications Group No. Treatment Route A 8μg IN B 24 μg IN C 80 μg IN D 240 μg IN E Flagella (Control) 240 μg IN F8 μg IM G 24 μg IM H 80 μg IM I 240 μg IM J Flagella (Control) 240 μg IMHumoral Immune Response

Escalating doses of Hexa-vaccine1 containing 8-240 μg of all theepitopes combined induced a specific humoral response to the H3N2 virus.The response in the groups immunized with the higher dose wassignificantly higher than the baseline. The change of titer between preimmune and immunized sera is shown in FIG. 6, which corresponds to thegroups in Table 5. A significant elevation over the pre-immune level(p<0.05) and over the control groups E and J that were immunized withnative flagella (IN or IM respectively in terms of specific recognitionof influenza virus H3N2 is observed in groups D and I that wereimmunized with 240 μg of Hexa-vaccine1 IN or IM, respectively.

Humoral Response: Specific Recognition of Flagella

The humoral response is also directed towards the flagella carrier. Thesera titer antibody to it is demonstrated in FIG. 7

FIG. 7 shows the humoral response to flagella following IN or IMadministration of Hexa-vaccine1. In the IN immunized groups (left handbars); there was an escalating dose response where the higher Abproduction was found where the higher dose (IN 240 μg) was given. In theIM administrated groups (right hand bars) a similarly high response wasobserved in all the doses ranging from 8 μg-240 μg/mouse. By bothroutes, the response to the native flagella is much higher; this mayresult from structural changes caused by the addition of foreignepitopes that may influence its immunogenicity.

Virus Titration

After a schedule of 3 vaccinations, the mice were infected with asub-lethal dose of influenza virus H3N2 strain (A/Texas/1/77). The lungsof the mice were removed 5 days later for titration of viral load inthem. The titration was performed in fertilized eggs FIG. 8). The viralload in the groups immunized with Hexa-vaccine1 (240 μg) is comparablein both IM and IN routes and is significantly lower (p<0.05) than thetiter in the control groups. This shows that Hexa-vaccine1 is effectiveand provides protection against virus infection.

Conclusions

Antibodies to (H3N2) virus: were significantly higher in the groupimmunized with the higher doses both IM and IN, but not in the groupsimmunized with lower doses, showing a dose escalating response.

Virus clearance: a lower virus titer was found in the groups immunizedIN and IM, while higher viral titers were detected in animals immunizedwith lower doses.

These data indicates that there is a correlation between dose andresponse, where only the higher doses led to significant protection.

No significant weight loss, behavioral abnormalities or signs forallergy (IgE elevation) were noticed as described in the following;

Example 10 IgE Response Following Immunization

For the evaluation of potential allergic response to our product, IgElevels were measured in all major experiments conducted in micefollowing immunization with different combination of epitopes or withnative flagella. In the various studies, mice were immunized IN or IMwith the recombinant flagella, total IgE in the sera was measured by acommercial murine IgE detection kit.

Low titers of <20 ng/ml were found in immunized animals similar to thenormal level in non immunized mice.

FIG. 9 presents the IgE concentration (ng/ml) in the dosing experimentand in the experiment for evaluating the cellular response. In both, IgEtiters on day 0 and after the third immunization were similar.

Mice were immunized either IN or IM with Hexa-vaccine1 at escalatingdoses, from 8 μg-240 μg/mouse. The sera from these mice was tested fortotal IgE

FIG. 10 represents the IgE concentration in sera of HHD transgenic (TG)mice immunized IN with recombinant flagella expressing influenzaepitopes.

Conclusions

In the sera samples from mice immunized with recombinant flagella, lowlevels of IgE were detected. The normal range for plasma IgE isestablished between 19 and 200 ng/ml using wild type animals). Titersobtained in immunized animal did not exceed 20 ng/ml and therefore,Hexa-vaccine does not induce allergic responses.

Example 11 Evaluation of Hexa-Vaccine2 in Rabbits

In this study rabbits were administered IN and IM with the vaccineproduct in a controlled environment at a Specific Pathogen Free (SPF)certified animal house. After a schedule of 3 vaccinations, bloodsamples were removed and specimens from the administration site andmajor organs were removed and subjected to histopathological analysis.

The study groups consisted of 3 immunizations comparing Hexa-vaccine2 (6epitopes: HA 91-108, HA 307-319, HA 354-372, NP 335-350, NP 380-393, M12-12) to PBS:

IN route: Hexa-vaccine2 100 μg and 50 μg/50 μl/rabbit

IM route: Hexa-vaccine2 600 μg and 300 μg/500 μl/rabbit

Neither mortality nor morbidity was observed in any of the groups.Histopathology results (2 weeks post immunization) showed:

Intranasal: No toxicity in the organs examined.

Intramuscular: No toxicity in the organs examined. One animal presentedfocal, minimal histolytic infiltrate at injection site.

Conclusions

Safety: The Hexa-vaccine2 was found to be safe and tolerable in rabbits.Humoral response: Ab titer specific to different influenza strains wasrecorded in some of the rabbits. It should be noted that distinctresponses were found between individual rabbits. FIG. 10 describes thefold increase in the antibody titer as compared to the pre immune titerin the responding rabbits. Non responding rabbits were excluded.

FIG. 11: Fold IgG titer to 3 different influenza strains in sera of NZWrabbits immunized IN 3 times with recombinant flagella expressing 6influenza epitopes.

Example 12 Pharmacokinetics

The pharmacokinetic studies revealed that the vaccine peak in sera wasobtained at 15 minutes and eliminated within 12 hours. In a parallelstudy with the vaccine formulated with adjuvant (Alum), the peakconcentration in sera was reached after 30 minutes and the vaccine waseliminated from the sera after 24 hours (data not shown). In addition,C_(max), T_(max), and AUC values were calculated as described hereunder.

Study Design

The study consisted of 10 groups of 3 males and 3 females per group thatwere administered with a single dose of 50 μg recombinantflagella/animal. Animals were bled at 10 predetermined time points of 5,10, 30 minutes and 1, 2, 4, 8, 12, 24 hours.

Pharmacokinetics Analysis

Calculation of the pharmacokinetic characteristics were based on theactual blood sampling time [h] (relative to the correspondingadministration time of Treatment) rounded to two decimal digits andnegative pre dose times set to zero. The sample before administrationwas used for calculation of the characteristics.

For calculation of the pharmacokinetic parameters, the following ruleswere applied:

Flagellin concentration values in sera at time points in the lag-timebetween time zero and the first quantifiable concentration wereconsidered as zero. Evaluation of relative bioavailability was performedfor the primary target parameters AUC and C_(max).

The log transformed values of the primary target parameters were subjectto an analysis of variance (ANOVA) model with the effects: sequence,subjects within sequence, period and treatment. The sequence effect wastested using the mean square of subjects within sequence from the ANOVAas an error term. All other effects were tested against the residualerror (error mean square) from the ANOVA. Based on the ANOVA 90%confidence intervals for the treatment ratios test*100/reference [%] wascalculated.

Individual treatment ratios test*100/reference [%] was given for theprimary target parameters. For T_(max) frequency tables were drawn bytreatment based on the nominal time of the T_(max) values.

FIG. 12 depicts protein serum concentration. Maximum serum concentration3,925 ng/ml (C_(max)) of Hexa-vaccine was observed after 15 minutes(T_(max)). Half (T_(1/2)) of the total exposure quantity was obtainedwithin 30 minutes post dosing. The area under the serumconcentration-time curve of 15,027 ng/ml indicates the body's totalexposure over time to Hexa-vaccine. No traces of protein could bedetected after 12 h.

Conclusions

A typical concentration curve was obtained at the end of thepharmacokinetics study with steep rise of the curve between 5 to 15minutes and moderate slope up until 12 hours. The maximum concentrationlevel (C_(max)=3,925 ng/ml) was observed upon 15 minutes, No traces ofprotein in the serum could be detected upon 12 hours post dosing. Theflagellin based vaccine will be totally eliminated from the sera within12 hours.

Epitope Safety

The selected conserved epitopes utilized in the Hexa-vaccine compriseepitopes that are restricted to the most prevalent HLA molecules inhuman. The selected epitopes are restricted to the viral structure andare not shared by any human protein therefore they are unlikely toinduce an autoimmune reaction.

While the present invention has been particularly described, personsskilled in the art will appreciate that many variations andmodifications can be made. Therefore, the invention is not to beconstrued as restricted to the particularly described embodiments, andthe scope and concept of the invention will be more readily understoodby reference to the claims, which follow.

REFERENCES

-   Ada G L and Jones P D, “The immune response to influenza infection”,    Curr. Topics Microbio. Immunol. 1986; 128:1.-   Anion R, Tarrab-Hazdai R, Ben-Yedidia T. Peptide-based synthetic    recombinant vaccines with anti-viral efficacy. Biologicals. 2001; 29    (3-4):237-42.-   Ben-Yedidia T, Marcus H, Reisner Y, Arnon R. Intranasal    administration of peptide vaccine protects human/mouse radiation    chimera from influenza infection. Int Immunol. 1999; 11 (7):1043-51.-   Gianfrani C, Oseroff C, Sidney J, Chesnut R, Sette A. Human memory    CTL response specific for influenza A virus is broad and    multispecific. Hum Immunol. 2000; 61:438-452.-   Ibrahim G F, Fleet G H, Lyons M J, Walker R A. Method for the    isolation of highly purified Salmonella flagellins. J Clin    Microbiol. 1985; (6):1040-4.-   Jeon S H, Ben-Yedidia T, Arnon R. Intranasal immunization with    synthetic recombinant vaccine containing multiple epitopes of    influenza virus. Vaccine. 2002; 20 (21-22):2772-80.-   Lamb R A, Zebedee S L, Richardson C D. Influenza virus M2 protein is    an integral membrane protein expressed on the infected-cell surface.    Cell. 1985; 40:627-633.-   Langer R. New methods of drug delivery. Science. 1990; 249    (4976):1527-33.-   Liu W, Zou P, Ding J, Lu Y, Chen Y H. Sequence comparison between    the extracellular domain of M2 protein human and avian influenza A    virus provides new information for bivalent influenza vaccine    design. Microbes Infect. 2005; 7 (2): 171-7.-   Meinkoth J, Wahl G. Hybridization of nucleic acids immobilized on    solid supports. Anal Biochem. 1984; 138 (2):267-84.-   O'Hagan D T, Jeffery H, Davis S S. Long-term antibody responses in    mice following subcutaneous immunization with ovalbumin entrapped in    biodegradable microparticles. Vaccine. 1993; 11 (9):965-9.-   Shapira M, Jolivet M, Arnon R. A synthetic vaccine against influenza    with built-in adjuvanticity. Int J Immunopharmacol. 1985; 7:719-723.-   Slepushkin V A, Katz J M, Black R A, Gamble W C, Rota P A, Cox N J.    Protection of mice against influenza A virus challenge by    vaccination with baculovirus-expressed M2 protein. Vaccine. 1995; 13    (15):1399-402.-   Townsend A R, Skehel J J. The influenza A virus nucleoprotein gene    controls the induction of both subtype specific and cross-reactive    cytotoxic T cells. J Exp Med. 1984; 160 (2):552-63.-   Zou P, Liu W, Chen Y H. The epitope recognized by a monoclonal    antibody in influenza A virus M2 protein is immunogenic and confers    immune protection. Int Immunopharmacol. 2005; 5 (4):631-5.

1. A vaccine for immunization of a subject comprising: (i) two influenzavirus peptide epitopes wherein the first peptide epitope is an influenzaA virus matrix (M) peptide epitope selected from the group consisting ofSEQ ID NOS: 25 and 26, and a second peptide epitope is a haemagglutinin(HA) peptide epitope as set forth in SEQ ID NO:48, and (ii) oneinfluenza B peptide epitope of a HA 354-372 peptide as set forth in SEQID NO:80, wherein the vaccine elicits cross strain protection.
 2. Thevaccine according to claim 1, wherein the M peptide epitope is an M1peptide epitope as set forth in SEQ ID NO:
 26. 3. The vaccine accordingto claim 1, wherein: (i) the M peptide epitope is the M1 2-12 (SEQ IDNO:25) peptide epitope; and (ii) the HA peptide epitope is HA 91-108(SEQ ID NO:48).
 4. The vaccine according to claim 3, further comprisinga CTL type NP peptide epitope as set forth in NP 335-350 (SEQ ID NO:67).5. The vaccine according to claim 1, further comprising an influenza Thelper (Th) type peptide epitope as set forth in HA 307-319 (SEQ IDNO:57).
 6. The vaccine according to claim 1, wherein: (i) the M peptideepitope is an influenza A virus M peptide epitope as set forth in M12-12 (SEQ ID NO:25); (ii) the HA peptide epitope is a HA 91-108 (SEQ IDNO:48) peptide epitope, with the vaccine further comprising: (iii) a Thtype peptide epitope as set forth in HA 307-319 (SEQ ID NO:57).
 7. Thevaccine according to claim 1, further comprising a CTL type NP peptideepitope as set forth in NP 335-350 (SEQ ID NO:67).
 8. The vaccineaccording to claim 1, further comprising an adjuvant or an excipient. 9.The vaccine according to claim 1, wherein each of the epitopes isexpressed individually within an expression vector.
 10. The vaccineaccording to claim 9, wherein each of the epitopes is expressedindividually within a recombinant flagellin.
 11. The vaccine accordingto claim 1, wherein each of the epitopes is expressed with a polypeptideselected from the group consisting of Cholera toxin, Tetanus toxin,Ovalbumin, Tuberculosis heat shock protein, Diphtheria Toxoid, Protein Gfrom respiratory syncytial virus, Outer Membrane Protein from Neisseriameningitides, nucleoprotein (N) of vesicular stomatitis virus,glycoprotein (G) of vesicular stomatitis virus, Plasmodium falciparumAntigen Glutamate-Rich Protein, Merozoite Surface Protein 3 or Virusesenvelope (E) protein.
 12. The vaccine according to claim 1, which isformulated for administration by a modality selected from the groupconsisting of intraperitoneal, subcutaneous, intranasal, intramuscular,oral, topical and transdermal delivery.
 13. A vaccine for immunizationof a subject comprises: (i) an influenza A virus matrix (M) peptideepitope as set forth in M1 2-12 (SEQ ID NO:25); (ii) HA 91-108 (SEQ IDNO:48); (iii) HA 307-319 (SEQ ID NO:57); (iv) NP 335-350 (SEQ ID NO:67);and (v) influenza B peptide epitope HA 354-372 (SEQ ID NO:80), whereinthe vaccine elicits cross strain protection.
 14. A method for elicitingan immune response and conferring protection against influenza virus ina subject, wherein the method comprises administering to the subject avaccine according to claim 1 to elicit an immune response and conferprotection against influenza virus.
 15. The method according to claim14, wherein the immune response is elicited against avian influenza,influenza A, influenza B or a combination thereof.